Method for augmenting the effects of serotonin reuptake inhibitors

ABSTRACT

A method of treating a subject for a serotonergic neurotransmission dysregulation disorder, comprises administering the subject a serotonin enhancer (e.g., a serotonin reuptake inhibitor) in an amount effective to treat the disorder; and concurrently administering the subject 5-hydroxytryptophan in an amount effective to enhance the activity of the serotonin enhancer, (e.g., serotonin reuptake inhibitor). In preferred embodiments the disorder is depression, anxiety, or substance abuse.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/397,806, filed Mar. 4, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/133,867, filed May 20, 2005, issued as U.S. Pat.No. 7,517,908, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/642,869 filed Jan. 11, 2005, and of U.S.Provisional Patent Application Ser. No. 60/642,800, filed Jan. 11, 2005,and of U.S. Provisional Patent Application Ser. No. 60/629,951, filedNov. 22, 2004, and of U.S. Provisional Patent Application Ser. No.60/606,811, filed Sep. 2, 2004, and of U.S. Provisional PatentApplication Ser. No. 60/573,265, filed May 21, 2004, the disclosures ofall of which are incorporated by reference herein in their entirety.

This application is related to Marc G. Caron, Xiaodong Zhang, MartinBeaulieu, Raul R. Gainetdinov, Tatiana D. Sotnikova, Ranga R. Krishnan,David A. Schwartz, Lauranell Burch, and Redford B. Williams,Polymorphism in Tryptophan Hydroxylase-2 Controls Brain SerotoninSynthesis, U.S. patent application Ser. No. 11/133,949, filed May 20,2005, and published as US 2006/0029951, the disclosure of which isincorporated by reference herein in its entirety.

GOVERNMENT FUNDING

This invention was made with Government support under grant numbers 5P50 MII60451 and 5 P50 MH60451 from the National Institute of MentalHealth. The United States Government has certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment of serotoninneurotransmission dysregulation disorders, including but not limited todepression, anxiety and substance abuse.

BACKGROUND OF THE INVENTION

Brain serotonin system plays a critical role in numerous neuronalfunctions and dysregulation of its homeostasis may contribute to manypsychiatric disorders (Veenestra-VanderWeele et al, 2000; Flattem andBlakely 2000; Blier and Abbott 2001; Tecott, 2003; Geyer, 1996;Tamminga, 1998; Snyder and Peroutka, 1982; Frazer, 1997; Aghajanian andMarek, 2000). In fact, numerous conditions such as unipolar majordepression and bipolar disorder, obsessive-compulsive disorder, anxiety,autism, personality disorder, panic and eating disorders, suicidality,chronic pain and post-traumatic stress syndrome and even attentiondeficit hyperactivity disorder (ADHD), are effectively treated byraising the extracellular concentrations of serotonin in the brain withcompounds including selective serotonin reuptake inhibitors (SSRIs) thatinhibit the neuronal re-uptake of serotonin. Like many otherneurotransmitters and modulators, the actions of serotonin are modulatedby the delicate balance between synthesis and degradation of thismonoamine. Tryptophan hydroxylase-1 (Tph1) has long been considered asthe sole rate-limiting enzyme for the synthesis of serotonin. However,Walther et al (2003) recently reported that inactivation of the Tph1gene in the mouse led to a decrease in the peripheral levels ofserotonin but no changes in central serotonin levels, suggesting thatanother form of the enzyme might exist. Data base mining yielded asecond related gene that was identified, cloned and named TPH2. Theproduct of the TPH2 gene is preferentially expressed in the brain asopposed to the predominant peripheral expression pattern of TPH1(Walther et al, 2003).

M. Bader et al., PCT Application WO 2004/007704 (US 2006/0275759),describes the identification of TPH2.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of treating asubject for a serotonergic neurotransmission dysregulation disorder,comprising: administering the subject a serotonin enhancer (e.g., aserotonin reuptake inhibitor) in an amount effective to treat thedisorder; and concurrently administering the subject 5-hydroxytryptophanin an amount effective to enhance the activity of the serotoninenhancer, (e.g., serotonin reuptake inhibitor). In preferred embodimentsthe disorder is depression, anxiety, or substance abuse.

A second aspect of the present invention is a method of treating asubject for a serotonergic neurotransmission dysregulation disorder,comprising: administering the subject a serotonin enhancer (e.g., aserotonin reuptake inhibitor) in an amount effective to treat thedisorder; and concurrently administering the subject a peripheraldecarboxylase inhibitor such as carbidopa in an amount effective toenhance the activity of the serotonin enhancer (e.g., selectiveserotonin reuptake inhibitor).

A third aspect of the invention is a method of treating a subject for aserotonergic neurotransmission dysregulation disorder, comprising:administering the subject a serotonin enhancer (e.g., a serotoninreuptake inhibitor) in an amount effective to treat said disorder; andconcurrently administering the subject a BH4 enhancer such astetrahydrobiopterin in an amount effective to enhance the activity ofthe serotonin enhancer (e.g., serotonin reuptake inhibitor).

A fourth aspect of the present invention is a method of treating asubject for a serotonergic neurotransmission dysregulation disorder,comprising: determining the presence or absence of at least one Tph2mutation in the subject; and then, if the subject possesses at least oneTph2 mutation; administering the subject 5-hydroxytryptophan in anamount effective to treat the disorder.

A fifth aspect of the present invention is a method of treating asubject for a serotonergic neurotransmission dysregulation disorder,comprising: determining the presence or absence of at least one Tph2mutation in the subject; and then, if the subject possesses at least oneTph2 mutation; administering the subject a peripheral decarboxylaseinhibitor in an amount effective to treat the disorder.

A sixth aspect of the present invention is a method of treating asubject for a serotonergic neurotransmission dysregulation disorder,comprising: determining the presence or absence of at least one Tph2mutation in said subject; and then, if said subject possesses at leastone Tph2 mutation; administering said subject a BH4 enhancer such astetrahydrobiopterin in an amount effective to treat said disorder.

In some embodiments of the foregoing, the disorder is depression,anxiety, or substance abuse; in other embodiments the disorder isanother serotonergic neurotransmission dysregulation disorder such asdescribed below.

A further aspect of the present invention is a pharmaceuticalformulation comprising, consisting of, or consisting essentially of:

(a) a serotonin enhancer such as a serotonin reuptake inhibitor;

(b) a serotonin precursor such as 5-hydroxytryptophan;

(c) optionally, but in some embodiments preferably, a peripheraldecarboxylase inhibitor such as carbidopa;

(d) optionally, but in some embodiments preferably, a BH4 enhancer suchas tetrahydrobiopterin; and

(e) a pharmaceutically acceptable carrier.

A further aspect of the present invention is a pharmaceuticalformulation comprising, consisting of, or consisting essentially of:

(a) a serotonin enhancer such as a serotonin reuptake inhibitor;

(b) a peripheral decarboxylase inhibitor such as carbidopa;

(c) optionally, but in some embodiments preferably, a BH4 enhancer suchas tetrahydrobiopterin;

(d) optionally, but in some embodiments preferably, a serotoninprecursor such as 5-hydroxytryptophan; and

(e) a pharmaceutically acceptable carrier.

A further aspect of the present invention is a pharmaceuticalformulation comprising, consisting of, or consisting essentially of:

(a) a serotonin enhancer such as a serotonin reuptake inhibitor;

(b) a BH4 enhancer such as tetrahydrobiopterin;

(c) optionally, but in some embodiments preferably, a peripheraldecarboxylase inhibitor such as carbidopa;

(d) optionally, but in some embodiments preferably, a serotoninprecursor such as 5-hydroxytryptophan; and

(e) a pharmaceutically acceptable carrier.

A further aspect of the present invention is a pharmaceuticalformulation comprising, consisting of, or consisting essentially of:

(a) a serotonin precursor such as 5-hydroxytryptophan;

(b) a peripheral decarboxylase inhibitor such as carbidopa;

(c) optionally, but in some embodiments preferably, a BH4 enhancer suchas tetrahydrobiopterin; and

(d) a pharmaceutically acceptable carrier.

A further aspect of the present invention is a pharmaceutical orneutraceutical formulation comprising, consisting of, or consistingessentially of:

(a) a serotonin precursor such as 5-hydroxytryptophan; and

(b) a BH4 enhancer such as tetrahydrobiopterin; and

(c) a pharmaceutically acceptable carrier; or in the case of aneutraceutical optionally other food ingredients such as fats, proteins,and/or carbohydrates.

A further aspect of the present invention is the use of5-hydroxytryptophan for the preparation of a medicament for carrying outa method of as described herein.

A further aspect of the present invention is the use of a peripheraldecarboxylase inhibitor such as carbidopa for the preparation of amedicament for carrying out a method as described herein.

A further aspect of the present invention is the use of a BH4 enhancersuch as tetrahydrobiopterin for the preparation of a medicament forcarrying out a method as described herein.

A further aspect of the present invention is the use of a serotoninenhancer such as a serotonin reuptake inhibitor for the preparation of amedicament for carrying out a method as described herein.

A still further aspect of the present invention is the use of a meansfor detecting a Tph2 mutation for carrying out a method as describedherein.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Serotonin levels in PC12 cells expressing R441H hTPH2 were lowerthan those in PC12 cells expressing WT hTPH2 (n=8 experiments).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

The disclosures of all United States Patent references cited herein areto be incorporated by reference herein in their entirety.

1. DEFINITIONS

“Genotyping” as used herein means determination of the type and numberof alleles present in a subject, whether determined by nucleic acidsequencing, PCR or RT-PCR amplification, examination of Tph2 protein, orany other method. A specific gene can be genotyped to determine if thegene is a wild-type or variant allele. Genotyping does not requiresequencing of the entire gene but may simply involve determining thepresence or absence of one or more mutations therein, as compared to the“wild type” gene.

“Tph2 mutation” as used herein refers to any mutation which leads toloss or decrease in activity of the encoded protein (tryptophanhydroxylase-2), such that the conversion of tryptophan to5-hydroxytryptophan in the subject by tryptophan hydroxylase-2 isreduced.

“Serotonin enhancer” as used herein refers to any compound thatincreases, directly or indirectly, the availability of serotonin in thecentral nervous system for binding to serotonin receptors at thepost-synaptic membrane, including but not limited to serotonin reuptakeinhibitors, monoamine oxidase inhibitors, tricyclic antidepressants,serotonin agonists, amphetamines, serotonin precursors, serotoninprodrugs, intermediates in the biosynthesis of serotonin, co-factors andpharmaceutically acceptable salts thereof. Such compounds may be givenalone or in combination with other serotonin enhancers.

“Serotonergic neurotransmission dysregulation disorder” as used hereinrefers to any disorder in which an increase or decrease in availableserotonin contributes, at least in part, to a disease, disorder, orcondition. Examples of such disorders include, but are not limited to,depressive disorder, anxiety disorder, social anxiety disorder,generalized anxiety disorder, bipolar disorder, schizophrenia, autism,epilepsy, mood disorders, alcohol or substance abuse and associateddisorders, panic disorder, migraine, obesity, bulimia, anorexia,premenstrual syndrome, menopause, sleep disorders,attention-deficit/hyperactivity disorder (ADHD), Tourette syndrome,aggression, obsessive compulsive disorder, pathological gambling,novelty seeking, borderline personality disorders, antisocialpersonality disorder, suicidality, eating disorders, sexual dysfunction,dementia, social phobia, fibromyalgia, overactive bladder, chronicfatigue syndrome, chronic pain, sudden infant death syndrome,post-traumatic stress syndrome, and Alzheimer's disease. These termshave their usual meaning in the art (see, e.g., DSM-IV).

“Depression” as used herein has its usual meaning in the art andincludes treating subjects with symptoms of depression and treatingsubjects diagnosed as depressed (e.g., subjects diagnosed as afflictedwith unipolar major depression)

“Unipolar major depression” (also called “unipolar depression” or “majordepression”) as used herein has its usual meaning in the art (see, e.g.,DSM-IV), and is typically characterized by the presence of at least fiveof the following symptoms for at least two weeks: Trouble sleeping orexcessive sleeping; a dramatic change in appetite, often with weightgain or loss; fatigue and lack of energy; feelings of worthlessness,self-hate, and inappropriate guilt; extreme difficulty concentrating;agitation, restlessness, and irritability; inactivity and withdrawalfrom usual activities; feelings of hopelessness and helplessness;recurring thoughts of death or suicide.

“Anxiety” as used herein has its conventional meaning in the art andincludes the treatment of subjects exhibiting symptoms of anxiety.

“Substance abuse” as used herein has its usual meaning and includes bothalcohol abuse or addiction (e.g., alcoholism, or alcoholic subjects), aswell as abuse or addiction to drugs such as narcotics, opiates,stimulants, depressants, etc. (e.g., barbiturates, ecstasy, cocaine,crack cocaine, morphine, heroin, amphetamine, methamphetamine,oxycontin, etc.).

Subjects for screening and/or treatment with the present invention are,in general, human subjects, but may also be other mammalian subjects(e.g., rodent subjects such as mouse or rat, primate subjects such ashuman or monkey, dog, cat, rabbit, etc.) for veterinary purposes.Subjects includes male and female subjects. The subject may be of anyrace and any age, including juvenile, adolescent, and adult.

“Treating” as used herein means the medical management of a subject,e.g., a human patient, with the intent to cure, ameliorate, stabilize,or prevent a disease, pathological condition, or disorder. This termincludes active treatment, that is, treatment directed specificallytoward the improvement or associated with the cure of a disease,pathological condition, or disorder, and also includes causal treatment,that is, treatment directed toward removal of the cause of theassociated disease, pathological condition, or disorder. In addition,this term includes palliative treatment, that is, treatment designed forthe relief of symptoms rather than the curing of the disease,pathological condition, or disorder; preventative treatment, that is,treatment directed to minimizing or partially or completely inhibitingthe development of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder. “Treating”also includes symptomatic treatment, that is, treatment directed towardconstitutional symptoms of the associated disease, pathologicalcondition, or disorder. Thus “treating” includes submitting orsubjecting a subject to a compound which will promote the elimination orreduction of a disease or symptoms of a disease, or which will slow theprogression of said disease. For example, a subject may be treated with,synthesized organic molecules, naturally occurring organic molecules,peptides, polypeptides, nucleic acid molecules, and components thereof“Treating” also includes the act of not giving a subject acontra-indicated therapeutic.

“Concurrent administration,” “administration in combination,”“simultaneous administration” or “administered simultaneously” as usedherein, interchangeably mean that the compounds are administered at thesame point in time or immediately following one another. In the lattercase, the two compounds are administered at times sufficiently closethat the results observed are indistinguishable from those achieved whenthe compounds are administered at the same point in time.

“Enhance the activity of” when used herein to describe the effect of asecond active agent on a first active agent means the second activeagent is administered in an amount that will (a) increase the treatmentefficacy of an otherwise efficacious active agent to an even moreefficacious level, as well as (b) increase the treatment efficacy of anotherwise ineffective active agent to an efficacious level. Tryptophanhydroxylase 2 (Tph2) is known and described at GenBank accession numbersNM_(—)173353 (human); NM_(—)173391 (mouse); and NM_(—)173839 (rat). Seealso M. Bader and D. Walther, PCT Patent Application WO 2004/007704 (US2006/0275759).

1. Tph2 Mutation or Polymorphism Detection/Genotyping.

In general, the step of detecting the polymorphism of interest, orgenotyping a subject, may be carried out by collecting a biologicalsample containing DNA from the subject, and then determining thepresence or absence of the polymorphism of interest in the biologicalsample. Any biological sample which contains the DNA of that subject maybe employed, including tissue samples and blood samples, with bloodcells being a particularly convenient source. The nucleotide sequence ofthe mouse and human Tph2 gene is known and suitable probes, restrictionenzyme digestion techniques, or other means of detecting thepolymorphism may be implemented based on this known sequence, or thevariations described herein, in accordance with standard techniques.See, e.g., U.S. Pat. Nos. 6,027,896 and 5,767,248 to A. Roses et al.

In describing the mutations disclosed herein in the novel proteinsdescribed herein, and the nucleotides encoding the same, the namingmethod is as follows: [amino acid replaced] [amino acid number insequence of known protein] [alternate amino acid]. For example, for themouse Tph2 variant disclosed herein, proline at the 447th amino acid inthe protein is replaced with arginine (Zhang, X et al., 2004).

The polymorphisms described herein can be detected in accordance withknown techniques based upon the known sequence information of the mouseand human Tph2 gene and the information provided herein. Novel nucleicacid sequences and proteins described herein can be isolated from humansources based upon the information provided herein or produced by othermeans such as site-directed mutagenesis of known or available aminoacids, coupled as necessary with techniques for the production ofrecombinant proteins known in the art.

Determining the presence or absence of DNA containing a polymorphism ormutation of interest may be carried out with an oligonucleotide probelabeled with a suitable detectable group, or by means of anamplification reaction such as a polymerase chain reaction or ligasechain reaction (the product of which amplification reaction may then bedetected with a labeled oligonucleotide probe or a number of othertechniques). Further, the detecting step may include the step ofdetecting whether the subject is heterozygous or homozygous for thepolymorphism of interest. Numerous different oligonucleotide probe assayformats are known which may be employed to carry out the presentinvention. See, e.g., U.S. Pat. No. 4,302,204 to Wahl et al.; U.S. Pat.No. 4,358,535 to Falkow et al.; U.S. Pat. No. 4,563,419 to Ranki et al.;and U.S. Pat. No. 4,994,373 to Stavrianopoulos et al. (applicantsspecifically intend that the disclosures of all U.S. Patent referencescited herein be incorporated herein by reference).

Amplification of a selected, or target, nucleic acid sequence may becarried out by any suitable means. See generally D. Kwoh and T. Kwoh,Am. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplificationtechniques include, but are not limited to, polymerase chain reaction,ligase chain reaction, strand displacement amplification (see generallyG. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); G.Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992)),transcription-based amplification (see D. Kwoh et al., Proc. Natl. Acad.Sci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or“3SR”) (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878(1990)), the Qβ replicase system (see P. Lizardi et al., BioTechnology6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or“NASBA”) (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), therepair chain reaction (or “RCR”) (see R. Lewis, supra), and boomerangDNA amplification (or “BDA”) (see R. Lewis, supra).

DNA amplification techniques such as the foregoing can involve the useof a probe, a pair of probes, or two pairs of probes which specificallybind to DNA containing the polymorphism of interest, but do not bind toDNA that does not contain the polymorphism of interest under the samehybridization conditions, and which serve as the primer or primers forthe amplification of the DNA or a portion thereof in the amplificationreaction. Such probes are sometimes referred to as amplification probesor primers herein.

In general, an oligonucleotide probe which is used to detect DNAcontaining a polymorphism or mutation of interest is an oligonucleotideprobe which binds to DNA encoding that mutation or polymorphism, butdoes not bind to DNA that does not contain the mutation or polymorphismunder the same hybridization conditions. The oligonucleotide probe islabeled with a suitable detectable group, such as those set forth belowin connection with antibodies. Such probes are sometimes referred to asdetection probes or primers herein.

Probes and primers, including those for either amplification and/orprotection, are nucleotides (including naturally occurring nucleotidessuch as DNA and synthetic and/or modified nucleotides) are any suitablelength, but are typically from 5, 6, or 8 nucleotides in length up to40, 50 or 60 nucleotides in length, or more. Such probes and or primersmay be immobilized on or coupled to a solid support such as a bead,chip, pin, or microtiter plate well in accordance with known techniques,and/or coupled to or labeled with a detectable group such as afluorescent compound, a chemiluminescent compound, a radioactiveelement, or an enzyme in accordance with known techniques.

Polymerase chain reaction (PCR) may be carried out in accordance withknown techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188. In general, PCR involves, first, treating anucleic acid sample (e.g., in the presence of a heat stable DNApolymerase) with one oligonucleotide primer for each strand of thespecific sequence to be detected under hybridizing conditions so that anextension product of each primer is synthesized which is complementaryto each nucleic acid strand, with the primers sufficiently complementaryto each strand of the specific sequence to hybridize therewith so thatthe extension product synthesized from each primer, when it is separatedfrom its complement, can serve as a template for synthesis of theextension product of the other primer, and then treating the sampleunder denaturing conditions to separate the primer extension productsfrom their templates if the sequence or sequences to be detected arepresent. These steps are cyclically repeated until the desired degree ofamplification is obtained. Detection of the amplified sequence may becarried out by adding to the reaction product an oligonucleotide probecapable of hybridizing to the reaction product (e.g., an oligonucleotideprobe of the present invention), the probe carrying a detectable label,and then detecting the label in accordance with known techniques, or bydirect visualization on a gel. When PCR conditions allow foramplification of all allelic types, the types can be distinguished byhybridization with allelic specific probe, by restriction endonucleasedigestion, by electrophoresis on denaturing gradient gels, or othertechniques.

Ligase chain reaction (LCR) is also carried out in accordance with knowntechniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general,the reaction is carried out with two pairs of oligonucleotide probes:one pair binds to one strand of the sequence to be detected; the otherpair binds to the other strand of the sequence to be detected. Each pairtogether completely overlaps the strand to which it corresponds. Thereaction is carried out by, first, denaturing (e.g., separating) thestrands of the sequence to be detected, then reacting the strands withthe two pairs of oligonucleotide probes in the presence of a heat stableligase so that each pair of oligonucleotide probes is ligated together,then separating the reaction product, and then cyclically repeating theprocess until the sequence has been amplified to the desired degree.Detection may then be carried out in like manner as described above withrespect to PCR.

It will be readily appreciated that the detecting steps described hereinmay be carried out directly or indirectly. For example, a polymorphismor mutation could be detected by measuring by digestion with restrictionenzymes, detection of markers that are linked to the mutation orpolymorphism, etc.

Genotype determinations can be compiled to predict either prognosis,drug efficacy, or suitability of a patient for participating in clinicaltrials of a neurological disease therapeutic. For example, the genotypemay be compiled with other patient parameters such as age, sex, diseasediagnosis, and known allelic frequency of a representative controlpopulation. A determination of the statistical probability of thepatient having a particular disease risk, drug response, or patientoutcome may be assessed from such genotype determinations. Patientoutcome, i.e. a prediction of a patient's likely health status, mayinclude a prediction of the patient's response to therapy,rehabilitation time, recovery time, cure rate, rate of diseaseprogression, predisposition for future disease, or risk of havingrelapse.

Kits useful for carrying out the methods of the present invention will,in general, comprise one or more oligonucleotide probes and otherreagents for carrying out the methods as described above, such asrestriction enzymes, optionally packaged with suitable instructions forcarrying out the methods.

Particular examples of Tph2 mutations that can be screen for ordetermined in carrying out the present invention include but are notlimited to mutations that encode a change in an amino acid of theencoded protein, said amino acid selected from the group consisting ofA65, V66, F68, L77, F84, I94, R97, E105, P152, W153, P155, D162, L175,R191, E211, V223, P244, G251, 8276, P277, V278, R285, 8294, P308, Y310,E313, A333, I339, A342, 5343, L344, A346, K353, V421, E423, A428, A436,R441, Y446, P449, Y450, and Q468. A mutation inducing any change in thenormal sequence (the amino acid to the left of the number identifyingthe amino acid location) is within the scope of the present invention(for example, a change of: I to T; E to G, P to Q; R to Q; G to V; P toL; R to W; Y to D; E to K; L to P; R to Q; Y to C; R to H; P to R; A toV; V to I; L to V; Q to X (where X is a stop codon) etc.).

Particularly preferred are the human P449R mutation, the human R441Hmutation the human W153R mutation, the human A65V mutation, the humanV661 mutation, the human L175V mutation, and the human Q468X (where X isa stop codon) mutation. Subjects may be determined to be heterozygous orhomozygous for the indicated mutation, or may be determined to carrydifferent mutations on the same, or different, alleles.

Note that 43 mutations are identified herein, some identified bysequence analysis and some by sequence identity compared to PAH. Table 1below lists corresponding mutations in Tph2 in six different species,which corresponding mutations are also useful for carrying out thepresent invention. It is striking that all 43 amino acids for thesemutations are virtually identical in Tph2 in six different species(except in two positions). This strongly indicates that mutations insimilar position in Tph2 have a like functional impact on serotoninproduction in other species.

TABLE 1 Corresponding mutations in additional species. human mouse ratchicken zebrafish pufferfish A65 A63 A60 A64 A35 A61 V66 V64 V61 V65 V36V62 F68 F66 F63 F67 F38 F64 L77 L75 L72 L76 L47 L73 F84 F82 F79 F83 F54F80 I94 I92 I89 I93 I64 I90 R97 R95 R92 R96 R67 R93 E105 E103 E100 E104E75 E101 P152 P150 P147 P151 P135 P148 W153 W151 W148 W152 W136 W149P155 P153 P150 P154 P138 P151 D162 D160 D157 D161 D145 D158 L175 L173L170 L174 L158 L171 R191 R189 R186 R190 R174 R187 E211 E209 E206 E210E194 E207 V223 V221 V218 V222 V206 V219 P244 P242 P239 P243 P227 P240G251 G249 G246 G250 G234 G247 R276 R274 R271 R275 R259 R272 P277 P275P272 P276 P260 P273 V278 V276 V273 V277 V261 V274 R285 R283 R280 R284R268 R281 R294 R292 R289 R293 R277 R290 P308 P306 P303 P307 P291 P304Y310 Y308 Y305 Y309 Y293 Y306 E313 E311 E308 E312 Y296 E309 A333 A331A328 A332 A316 A329 I339 I337 I334 I338 I322 I335 A342 A340 A337 A341A325 A338 S343 S341 S338 S342 S326 S339 L344 L342 L339 L343 L327 L340A346 A344 A341 A345 A329 A342 K353 K351 K348 K352 K336 K349 V421 V419V416 V420 V404 V417 E423 E418 E422 E406 E419 A428 A426 A423 A427 A411A424 A436 A434 A431 A435 A419 A432 R441 R439 R436 R440 R424 R437 Y446Y444 Y441 Y445 Y429 Y442 P449 P447 P444 P448 P432 P445 Y450 Y448 Y445Y449 Y433 Y446 Q468 Q466 Q463 Q467 Q464

In addition to the foregoing, intronic mutations are useful for carryingout the present invention. For example, a G->A mutation at position 144in Intron 6 of human TPH2 is useful for carrying out the presentinvention. This is a non-coding mutation in the Intron 6 and ispredicted to cause an alternative splicing variant/mutation of humanTPH2. The entire intron 6 in human TPH2 is 6236 base pairs in length;the pertinent portion of exon 6 is shown in panel a of Table 2 below,and the pertinent portion of the mutant intron 6 is shown in panel B ofTable 2 below.

TABLE 2  Intron 6 non-coding mutation. AGGTCAGCCCATTCCCAGGGTGGAGTATACTGAAGAAGAAACTAAAACTTGGGGTGTTGTATTCCGGGAGCTCTCCAAACTCTATCCCACTCATGCTTGCCGAGAGTATTTGAAAAACTTCCCTCTGCTGACTAAATACTGTGGCTACAGAGAGGACAATGTGCCTCAACTCGAAGATGTCTCCATGTTTCTGAAAG (SEQ ID NO: 1) Bgtaagatttcacacaggctgtctcttattagtcaatatcctcaattgccttccaaggacacaggttgcagcaatggctctttttccaaaaaaggaaaaacagtgatttaaaaaattgttggctttgagc caacaattacctgc ggccacctgtgggaagcagagcaagggac tcagctgcttttgcagctcaggagcttgctgaggcctctttgtggctggttgttgtaaatggtaaggcccaaaggatatttgcaagttcagctctgagctttttctgatccaggagctgctgtgctgggctacatgagtatgaaatgacctccaaaagtgcctttttatttgctttgttaaaaagtat (SEQ ID NO: 2) The g/a polymorphism is shown in boldunderlined font in panel b.

2. ACTIVE AGENTS

Active agents used to carry out the present invention are, in general,serotonin enhancers. Numerous serotonin enhancers and serotonin enhancertherapies are known. See, e.g., U.S. Pat. No. 6,218,395.

The serotonin enhancer can be a serotonin reuptake inhibitor orselective serotonin reuptake inhibitor, such as described in U.S. Pat.Nos. 6,552,014; 6,492,366; 6,387,956; 6,369,051; or 5,958,429. Examplesof known serotonin reuptake inhibitors that may be used in carrying outthe present invention include, but are not limited to:

-   -   cianopramine or a pharmaceutically acceptable salt thereof        (e.g.,        5-[3-(dimethylamino)propyl]-10,11-dihydro-5H-dibenz[b,f]azepine-3-carbonitrile);    -   citalopram or a pharmaceutically acceptable salt thereof (e.g.,        1-[3-(dimethylamino)propyl]-1-(p-fluorophenyl)-1,3-dihydro-isobenzofuran-5-carbonitrile);    -   escitalopram or a pharmaceutically acceptable salt thereof        (e.g.,        (S)-1-3-dimethylamino-propyl-1-(4′-fluoro-phenyl)-1,3-dihydro-isobenzofuran-5-carbonitril,        oxalate);    -   dapiprazole or a pharmaceutically acceptable salt thereof (e.g.,        5,6,7,8-tetrahydro-3-[2-(4-o-tolyl-1-piperazinyl)ethyl]-1,2,4-triazolo[4,3-a]pyridine        (hydrochloride));    -   desvenlafaxine or a pharmaceutically acceptable salt thereof        (e.g., Phenol,        4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl]-(Z)-2-butanedioate        (1:1) monohydrate);    -   duloxetine or a pharmaceutically acceptable salt thereof,        including LY 223.332, LY264.452 ((−)-enantiomer), LY248.686        (HCl), and Y227.942 (+)-enantiomer) (e.g.,        (+)-(S)—N-methyl-1-(1-naphtyloxy)-2-thiophenepropylamine (HCl));    -   fluoxetine or a pharmaceutically acceptable salt thereof (e.g.,        3-[(p-trifluoromethyl)phenoxy]-N-methyl-3-phenyl-propylamine        (hydrochloride));    -   fluvoxamine or a pharmaceutically acceptable salt thereof (e.g.,        (E)-5-methoxy-4′-(trifluoromethyl)valerophenone        O-(2-amino-ethyl)oxime (hydrogen maleate));    -   ifoxetine or a pharmaceutically acceptable salt thereof (e.g.,        (+/−)-cis-4-(2,3-xylyloxy)-3-piperidinol (sulfate));    -   indalpine or a pharmaceutically acceptable salt thereof (e.g.,        3-[2-(4-piperidyl)ethyl]indole);    -   LY 113.821 or a pharmaceutically acceptable salt thereof (e.g.,        N-methyl-3-(1-naphthoxy)-3-phenylpropylamine);    -   mirtazapine or a pharmaceutically acceptable salt thereof (e.g.,        1,2,3,4,10,14-hexahydro-2        methylpiprazino[2,1-a]pyrido[2,3-c]benzazepine);    -   nefazodone or a pharmaceutically acceptable salt thereof (e.g.,        1-[3-[4-(m-chlorophenyl)-1-piperazinyl]propyl]-3-ethyl-4-(2-phenoxyethyl)-²-1,2,4-triazolin-5-one        (HCl));    -   2-nitroimipramine or a pharmaceutically acceptable salt thereof        (e.g.,        5-[3-(dimethylamino)propyl]-2-nitro-10.11-dihydro-5H-dibenz[b,f]azepine        (hydrochloride);    -   nortriptyline or a pharmaceutically acceptable salt thereof        (e.g.,        10.11-dihydro-N-methyl-5H-dibenzo[a,d]cycloheptene-Δ^(5,Γ)propylamine        (hydrochloride));    -   paroxetine or a pharmaceutically acceptable salt thereof (e.g.,        (3S-trans)-3-[(1,3-benzodioxol-5-yloxy)        methyl]-4-(4-fluorophenyl)piperidine));    -   RU 25.591 or a pharmaceutically acceptable salt thereof (e.g.,        cis-6,7,8,9-tetrahydro-N,N-dimethyl-5-(p-nitrophenoxy)-5H-benzocyclohepten-7-amine        (fumarate));    -   sercloremine or a pharmaceutically acceptable salt thereof        (e.g., 4-(5-chloro-2-benzofuranyl)-1-methylpiperidine        (hydrochloride));    -   sertraline or a pharmaceutically acceptable salt thereof (e.g.,        (+)-cis(1S,        4S)-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1-naphthylamine        (HCl));    -   setiptiline or a pharmaceutically acceptable salt thereof (e.g.,        2,3,4,9-tetrahydro-2-methyl-1H-dibenzo[c,f]cyclohepta[1,2-c]pyridine        (maleate));    -   tianeptine or a pharmaceutically acceptable salt thereof (e.g.,        N-(3-chloro-6,11-dihydro-6-methyldibenzo[c,f][1,2]thiazepin-11-yl)-7-amino-heptanoic        acid S, S-dioxide);    -   trazodone or a pharmaceutically acceptable salt thereof (e.g.,        2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazolo[4,3-a]pyridin-3(2H)-one        (hydrochloride));    -   venlafaxine or a pharmaceutically acceptable salt thereof (e.g.,        (+/−)-1-[2-(dimethylamino)-1-(p-methoxyphenyl)ethyl]cyclohexan-1-ol        (HCl))    -   viqualine or a pharmaceutically acceptable salt thereof (e.g.,        cis-6-methoxy-4-[3-(3R,4R)-(3-vinylpiperidyl)        propyl]-quinoline); and    -   zimeldine or a pharmaceutically acceptable salt thereof (e.g.,        (Z)-3-(p-bromophenyl)-N,N-dimethyl-3-(3-pyridyl)allylamine).

The serotonin enhancer can be a monoamine oxidase inhibitor such asdescribed in U.S. Pat. Nos. 6,472,423 and 6,011,054. Examples ofmonoamine oxidase inhibitors include but are not limited toIsocarboxazid, phenelzine, and tranylcypromine. The serotonin enhancercan be a serotonin agonist such as described in U.S. Pat. Nos.6,656,172; 6,579,899 and 6,387,907. The serotonin enhancer can be anamphetamine (including derivatives thereof such as phentermine,fenfluramine, and (+)-3,4-methylenedioxyamphetamine. The serotoninenhancer can be a tricyclic antidepressant such as described in U.S.Pat. Nos. 6,368,814; 6,358,944; 6,239,162; and 6,211,171. Examples oftricyclic antidepressants include but are not limited to imipramine,amitriptyline and clomipramine.

The serotonin enhancer can be an anxiolytic such as buspirone oripsapirone.

The serotonin enhancer can be a precursor or prodrug of serotonin, or anintermediate in serotonin biosynthesis, such as described in U.S. Pat.Nos. 6,579,899; 6,013,622; and 5,595,772. Examples includes tryptophan,5-hydroxytryptophan, TPH2 co-factor tetrahydrobiopterin and itsprecursors, a tryptophan-rich diet or dietary supplements of tryptophan.

Decarboxylase inhibitors, particularly peripheral decarboxylaseinhibitors, useful for carrying out the present invention includecarbidopa (particularly L-carbidopa) and/or benserazide. See, e.g., U.S.Pat. No. 6,387,936.

Tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (or“BH4”), useful as an active agent in the some embodiments of the presentinvention, is known. See, e.g., U.S. Pat. Nos. 6,451,788 and 4,920,122.While the present invention is described primarily with reference toBH4, other BH4 enhancers such as folates and analogs thereof, (e.g.,methylfolate) as well as estrogen agonists and glucocorticoidantagonists may be used in addition, or in alternative, thereto. Folateanalogs are known and described in, for example, U.S. Pat. Nos.6,808,725; 6,673,381; 6,500,829; and 6,191,133.

3. PHARMACEUTICAL FORMULATIONS

The active compounds described above may be formulated alone or incombination with each other (e.g., in the combinations described above)for administration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995).

In the manufacture of a pharmaceutical formulation according to theinvention, the active compound (including the physiologically acceptablesalts thereof) is typically admixed with, inter alia, an acceptablecarrier. The carrier must, of course, be acceptable in the sense ofbeing compatible with any other ingredients in the formulation and mustnot be deleterious to the patient. The carrier may be a solid or aliquid, or both, and is preferably formulated with the compound as aunit-dose formulation, for example, a tablet, which may contain from0.01 or 0.5% to 95% or 99% by weight of the active compound orcompounds. One or more active compounds may be incorporated in theformulations of the invention, which may be prepared by any of the wellknown techniques of pharmacy comprising admixing the components,optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a active agent, ina unit dosage form, in a sealed container. The compound or salt isprovided in the form of a lyophilizate which is capable of beingreconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection thereof into a subject.The unit dosage form typically comprises from about 10 mg to about 10grams of the compound or salt. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agentwhich is physiologically acceptable may be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

For aerosol delivery to the airway surfaces of a patient thepharmaceutical (e.g., as a dry powder or as liquid particles) theformulation may be prepared in aerosolizable form and provided in anaerosol or inhalation delivery device.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6)318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2Mactive ingredient.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the compounds disclosedherein or salts thereof, may be lyophilized to produce a lyophilizatewhich may be reconstituted with a pharmaceutically acceptable carrier,such as water, to regenerate a liposomal suspension.

Other pharmaceutical compositions may be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

In addition to active compounds or their salts, the pharmaceuticalcompositions may contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the compositions may contain microbialpreservatives. Useful microbial preservatives include methylparaben,propylparaben, and benzyl alcohol. The microbial preservative istypically employed when the formulation is placed in a vial designed formultidose use. Of course, as indicated, the pharmaceutical compositionsof the present invention may be lyophilized using techniques well knownin the art.

The compositions of this invention can also be administered as a foodbased preparation, functional food, dietary supplement or nutraceutical.For the purposes of this application, “functional food” is defined as afood engineered or supplemented to give improved nutritional value,“dietary supplement” is defined as a substance produced by isolation, ormicrobial culture purification that gives health benefits, and“nutraceutical” is defined as a food, or parts of a food, that providemedical or health benefits, including prevention and treatment ofclinical conditions and/or symptoms related thereto.

Active agents may be included in a pharmaceutical formulation in anamount ranging from 0.001, 0.01, 0.1 or 1 percent by weight, up to about10, 20, 50 percent by weight, or more, depending upon the activity ofthe particular active agent. For example, in some embodiments apharmaceutical formulation of the invention comprises, consists of, orconsists essentially of:

-   -   (a) from 0.001, 0.01 or 0.1 to 20 or 40 percent by weight of a        serotonin enhancer such as a serotonin reuptake inhibitor;    -   (b) from 0.1 or 1 to 40 or 60 percent by weight of a serotonin        precursor such as 5-hydroxytryptophan;    -   (c) optionally, 0.001, 0.01 or 0.1 to 20 or 40 percent by weight        of a peripheral decarboxylase inhibitor such as carbidopa;    -   (d) optionally, from 0.1 or 1 to 40 or 60 percent by weight of a        BH4 enhancer such as tetrahydrobiopterin; and    -   (e) from 1 to 99.9 percent by weight of a pharmaceutically        acceptable carrier.        In some other exemplary embodiments, a pharmaceutical        formulation of the invention comprises, consists of, or consists        essentially of:    -   (a) from 0.001, 0.01 or 0.1, up to 20 or 40 percent by weight of        a serotonin enhancer such as a serotonin reuptake inhibitor;    -   (b) from 0.001, 0.01 or 0.1, up to 20 or 40 percent by weight of        decarboxylase inhibitor such as carbidopa;    -   (c) optionally, from 0.1 or 1 to 40 or 60 percent by weight of a        BH4 enhancer such as tetrahydrobiopterin;    -   (d) optionally, from 0.1 or 1 to 40 or 60 percent by weight of a        serotonin precursor such as 5-hydroxytryptophan; and    -   (e) from 1 to 99.9 percent by weight of a pharmaceutically        acceptable carrier.        In some other exemplary embodiments a pharmaceutical formulation        of the invention comprises, consists of, or consists essentially        of    -   (a) 0.001, 0.01 or 0.1 to 20 or 40 percent by weight of a        serotonin enhancer such as a serotonin reuptake inhibitor;    -   (b) from 0.1 or 1 to 40 or 60 percent by weight of a BH4        enhancer such as tetrahydrobiopterin;    -   (c) optionally, 0.001, 0.01 or 0.1 to 20 or 40 percent by weight        of a peripheral decarboxylase inhibitor such as carbidopa;    -   (d) optionally, from 0.1 or 1 to 40 or 60 percent by weight of a        serotonin precursor such as 5-hydroxytryptophan; and    -   (e) from 1 to 99.9 percent by weight of a pharmaceutically        acceptable carrier.        In other exemplary embodiments, a pharmaceutical formulation of        the invention comprises, consists of, or consists consisting        essentially of:    -   (a) from 0.1 or 1 to 40 or 60 percent by weight of a serotonin        precursor such as 5-hydroxytryptophan; and    -   (b) from 0.001, 0.01 or 0.1, up to 20 or 40 percent by weight of        decarboxylase inhibitor such as carbidopa;    -   (c) optionally, from 0.1 or 1 to 40 or 60 percent by weight of a        BH4 enhancer such as tetrehydrobiopterin;    -   (d) from 1 to 99.9 percent by weight of a pharmaceutically        acceptable carrier.        And in still other embodiments, a pharmaceutical (this term        including neutraceutical) formulation of the invention        comprises, consists of or consists essentially of:    -   (a) from 0.1 or 1 to 40 or 60 percent by weight of a serotonin        precursor such as 5-hydroxytryptophan; and    -   (b) from 0.1 or 1 to 40 or 60 percent by weight of a BH4        enhancer such as tetrahydrobiopterin; and    -   (c) from 1 to 99.9 percent by weight of a pharmaceutically        acceptable carrier (in the case of a neutraceutical optionally        from 1 to 99.9 percent by weight of other food ingredients such        as fats, proteins, carbohydrates, and combinations thereof).

Such formulations may be provided in any suitable form including thosedescribed above, such as tablets, capsules, suppositories, inhalation oraerosolizable formulations, formulations in an inhalation deliverydevice, parenterally injectable formulations. etc. In the case of aneutraceutical composition, the formulation may be provided in the formof a bar, beverage, drink, snack food, etc.

4. SUBJECTS, DOSAGE AND ROUTES OF ADMINISTRATION

Subjects to be treated by the methods of the invention are, in someembodiments (such as those involving administration of a serotoninenhancer such as a serotonin reuptake inhibitor in combination with aserotonin precursor and/or a peripheral decarboxylase inhibitor) thoseexhibiting symptoms of a serotonin neurotransmission dysregulationdisorder (particularly depression, anxiety, or substance abuse, althoughit will be understood that subjects afflicted with other serotoninneurotransmission dysregulation disorders such as set forth above may betreated as well). Subjects may be those previously determined to benon-responsive or insufficiently responsive to treatment therapy with aserotonin enhancer such as a serotonin reuptake inhibitor alone (e.g.,subjects who exhibited no benefit or improvement in symptoms toadministration of a serotonin enhancer such as a serotonin reuptakeinhibitor, or subjects who exhibited insufficient benefit or improvementin symptoms to administration of a serotonin enhancer such as aserotonin reuptake inhibitor).

Subjects may, in some preferred embodiments, be genotyped or determinedto carry a Tph2 mutation prior to initiation of the treatments describedherein. Such subjects may be more confidently administered thetreatments described herein based on a greater expectation of likelyclinical benefit of these treatments, based on the greater understandingof the underlying genetic and physiological basis of the disorder asdescribed herein.

The active compounds may be administered to the subject by any suitableroute, including those indicated with respect to particular formulationsabove. Thus suitable routes of administration include oraladministration, buccal administration, parenteral injection, inhalationor aerosol administration, transcutaneous administration, etc.

The therapeutically effective dosage of any specific active compound,the use of which is in the scope of present invention, will varysomewhat from compound to compound, and patient to patient, and willdepend upon the condition of the patient and the route of delivery.

For example, a serotonin enhancer such as a serotonin reuptake inhibitorcan be administered to the average adult human for the treatment of thedisorders described herein in an amount of from about 0.1 mg to about2000 mg, preferably from about 1 mg to about 200 mg per unit dose.

A peripheral decarboxylase inhibitor such as carbidopa or benserazidecan, in some embodiments, be administered to an average adult humansubject in an amount of from 5 or 10 to 25, 50 or 100 milligrams perunit dose.

A precursor or prodrug of serotonin, or an intermediate in serotoninbiosynthesis, such as 5-hydroxytryptophan, may be administered in anamount of from about 1, 2 5 or 10 milligrams up to 0.1, 0.5, 1 or 4grams per unit dose.

Administration of unit doses of active agents, alone or in combination,may be one or several times daily, for example 2, 3, 4 or 8 times,giving for example, 1, 2 or 3 doses each time.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

TPH1 and 2 convert tryptophan to 5-hydroxytryptophan, which is thendecarboxylated by aromatic amino acid decarboxylase (AADC) to serotonin.The two enzymes belong to the family of aromatic amino acidhydroxylases. Members of this family include phenylalanine hydroxylase(PAH) and tyrosine hydroxylase (TH) respectively involved in thesynthesis of tyrosine and L-Dopa, the precursor of dopamine. Both TH andTPH catalyze the rate limiting steps in the synthesis of dopamine andserotonin. There is a high degree of sequence conservation in the genesfor these four different enzymes. These enzymes show considerablestructural similarities and require the same co-factor for function(Fitzpatrick, 1999). Over 400 mutations have been previously identifiedin the PAH gene and these mutations, which result in reduced enzymeactivity, produce hyperphenylalaninemia causing of one of the mostprevalent condition identified in newborns, phenylketonuria (PKU)(Scriver et al, 2003).

A (G1463A) SNP which replaces a highly conserved Arg441 (CGT) with His441 (CAT) has been identified resulting in ˜80% decrease in serotoninlevels as compared to wild type TPH2 when expressed in PC12 cells (FIG.1), indicating a severe loss-of-function phenotype of TPH2 (R441H) inserotonin synthesis. In addition, allele-specific genotyping hasidentified that 9 subjects were found to carry the 1463A allele in acohort of 87 subjects diagnosed with unipolar major depression, whileonly 3 subjects from a cohort of non-unipolar major depression (orcontrol) subjects (N=279) were found to carry the 1463A allele (Table3). The presence of this functional SNP in unipolar major depressionsubjects versus non-unipolar major depression subjects was statisticallysignificant (P=1.989E-04 by Fisher's Exact Test). Moreover, among these9 unipolar major depression subjects, 7 had family history of mentalillness or drug and alcohol abuse, 6 had suicidal ideation or attemptand 4 had generalized anxiety symptoms. Interestingly, 7 subjectsexhibited lack of responsiveness to SSRI, while 2 subjects wereresponsive only to high doses of SSRI (Table 3). However, among the 3non-unipolar major depression (or control) subjects carrying 1463Aallele, one subject had generalized anxiety symptoms, while the othertwo had mild depression and family history of mental illness or drug andalcohol abuse (Table 3), indicating a higher susceptibility for certainneuropsychiatric disorders in the presence of 1463A allele. Therefore,identification of a loss-of-function mutation in hTPH2 provides directevidence for a role of brain serotonin synthesis in unipolar majordepression.

TABLE 3 Summary of genotype and history of subjects. Patient Family SSRII.D. Sex Age Allele History Suicidality Anxiety Response Note UnipolarMajor Depression Patients N = 87 1202 F 72 A/A − + − + Sertraline 200 mg1294 M 80 G/A + + − − 1496 M 74 A/A − − + + Sertraline 100 mg 1745 M 71G/A + − + − 1747 M 82 G/A + + − − 1839 F 69 G/A + − − − 1851 F 65G/A + + − − 1902 F 77 A/A + + + − 1975 M 64 G/A + + + − Non-UnipolarMajor Depression Subjects N = 279 1174 A/A − + 1541 G/A + − Milddepression 1996 G/A + − Mild depression Family history: family historyfor mental illness, drug or alcohol abuse Suicidality: suicidal ideationor attempt Anxiety: three or more anxiety symptoms

REFERENCES

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The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of treating a subject for aserotonergic neurotransmission dysregulation disorder, comprising:administering said subject a serotonin reuptake inhibitor in an amounteffective to treat said disorder; and concurrently administering saidsubject 5-hydroxytryptophan in an amount effective to enhance theactivity of said serotonin reuptake inhibitor; wherein said subject isnon-responsive or insufficiently responsive to treatment therapy with aserotonin reuptake inhibitor alone.
 2. The method of claim 1, whereinsaid subject has been predetermined to have at least one Tph2 mutation.3. The method of claim 2, wherein said Tph2 is human Tph2 and saidmutation encodes a change in an amino acid of the encoded protein, saidamino acid selected from the group consisting of A65, V66, F68, L77,F84, I94, R97, E105, P152, W153, P155, D162, L175, R191, E211, V223,P244, G251, R276, P277, V278, R285, R294, P308, Y310, E313, A333, I339,A342, S343, L344, A346, K353, V421, E423, A428, A436, R441, Y446, P449,Y450, and Q468.
 4. The method of claim 2, wherein said Tph2 mutation isselected from the group consisting of the human P449R mutation, thehuman R441H mutation, the human W153R mutation, the human A65V mutation,the human V66I mutation, the human L175V mutation, and the human Q468X(where X is a stop codon) mutation.
 5. The method of claim 1, whereinsaid serotonin reuptake inhibitor is selected from the group consistingof citalopram, fluvoxamine, fluoxetine, sertraline, paroxetine,escitalopram, duloxetine, venlafaxine, and pharmaceutically acceptablesalts thereof.
 6. The method of claim 1, wherein said disorder isdepression.
 7. The method of claim 1, wherein said disorder is anxiety.8. The method of claim 1, wherein said disorder is unipolar majordepression.
 9. The method of claim 1, wherein said disorder is bipolardisorder.
 10. The method of claim 1, wherein said disorder is obesity.11. The method of claim 1, wherein said subject is a human subject. 12.A method of treating a subject for a serotonergic neurotransmissiondysregulation disorder, wherein said subject is being treated with aserotonin reuptake inhibitor, comprising: determining whether thesubject is non-responsive or insufficiently responsive to treatmenttherapy with a serotonin reuptake inhibitor alone; and then, if saidsubject is non-responsive or insufficiently responsive to said serotoninreuptake inhibitor; administering said subject 5-hydroxytryptophan in anamount effective to enhance the effects of said serotonin reuptakeinhibitor to treat said disorder.
 13. The method of claim 12, whereinsaid subject has been predetermined to have at least one Tph2 mutation.14. The method of claim 12, wherein said serotonin reuptake inhibitor isa selective serotonin reuptake inhibitor.
 15. The method of claim 12,wherein said serotonin reuptake inhibitor is italopram, fluvoxamine,fluoxetine, sertraline, paroxetine, escitalopram, duloxetine, orvenlafaxine, or a pharmaceutically acceptable salt thereof.
 16. Themethod of claim 12, wherein said disorder is depression.
 17. The methodof claim 12, wherein said disorder is anxiety.
 18. The method of claim12, wherein said disorder is unipolar major depression.
 19. The methodof claim 12, wherein said disorder is bipolar disorder.
 20. The methodof claim 12, wherein said disorder is obesity.
 21. The method of claim12, wherein said subject is a human subject.